Δx0 = 0.38 nm and a zero-force off-rate koff,0 in the 10−6 s-1 range.The widely used interaction of the homotetramer streptavidin with the small molecule biotin has been intensively studied by force spectroscopy and has become a model system for receptor ligand interaction. However, streptavidin’s tetravalency results in diverse force propagation pathways through the different binding interfaces. This multiplicity gives rise to polydisperse force spectroscopy data. Here, we present an engineered monovalent streptavidin tetramer with a single cysteine in its functional subunit that allows for site-specific immobilization of the molecule, orthogonal to biotin binding. Functionality of streptavidin and its binding properties for biotin remain unaffected. We thus created a stable and reliable molecular anchor with a unique high-affinity binding site for biotinylated molecules or nanoparticles, which we expect to be useful for many single-molecule applications. To characterize the mechanical properties of the bond between biotin and our monovalent streptavidin, we performed force spectroscopy experiments using an atomic force microscope. We were able to conduct measurements at the single-molecule level with 1:1-stoichiometry and a well-defined geometry, in which force exclusively propagates through a single subunit of the streptavidin tetramer. For different force loading rates, we obtained narrow force distributions of the bond rupture forces ranging from 200 pN at 1,500 pN/s to 230 pN at 110,000 pN/s. The data are in very good agreement with the standard Bell-Evans model with a single potential barrier at  For single-molecule techniques, the tetravalency of SA can however be disadvantageous, as it promotes clustering of biotinylated molecules. Single-molecule force spectroscopy (SMFS) [With its low dissociation constant in the femtomolar range , its spey (SMFS) , super-ry (SMFS)  often rey (SMFS) . Howevery (SMFS) , monomery (SMFS) . In 2006y (SMFS)  developeThe interaction between biotin and tetravalent SA/avidin was the first receptor-ligand interactions probed by AFM-based SMFS –19. It hIn 1994, Moy et al. [Several groups independently repeated the experiment , 21. AllDue to different ways the ligand binds to the receptor, AFM-based SMFS data can be dispersed when performing experiments using multivalent receptor molecules, such as SA, even if actual single-molecule interactions are probed. Pulling on the ligand, the force can propagate through the receptor molecule in different ways . This reFrom the crystal structure of wild-type SA, it can be reasoned that the SA monomers assemble into strongly associated dimers that form less stable tetramers . TherefoNon-equilibrium unbinding forces are loading rate dependent . Any comWe therefore implement mSA to perform high-throughput AFM-based SMFS experiments for probing the mechanical stability of the biotin:SA system in a well-defined pulling geometry, no longer distorted by the receptor’s multivalency. The quality of the data is further improved by the use of protein calibration domains for identification of single interactions. The unfolding patterns of the calibration domains that are enzymatically fused to ligand or receptor molecule verify single rupture events. When unfolding under the applied load before the receptor-ligand complex ruptures, they yield a specific unfolding force, which serves as internal reference for force calibration, and a defined length increment that is taken as an indicator for single receptor-ligand unbinding.For site-selective immobilization of SA, we genetically modified the functional subunit of mSA. Although wildtype SA does not contain any cysteine residues, the SA tetramer was found to be of high stability under conditions, which are usually denaturing . In contE. coli BL21(DE3)-CodonPlus . The constructs formed inclusion bodies that were isolated as described previously [A detailed description of expression and purification is provided in the supplement . SA and eviously . To recoDictyostelium discoideum fourth filamin domain (ddFLN4) construct with an N-terminal ybbR-tag [E. coli BL21(DE3)-CodonPlus  and lysis, purification was achieved by immobilized metal ion affinity chromatography (Ni-IMAC).The ybbR-tag  and a C-E. coli BL21(DE3)-CodonPlus . Purification was performed by Ni-IMAC.The superfolder green fluorescent protein (GFP) construct with an N-terminal cysteine and a C-terminal ybbR-tag was cloned into pET vectors  and expressed in 2 and 50 mM HEPES at pH 7.5 for 1 h at 37°C. To clean the solution from remaining CoA-Biotin, a buffer exchange to phosphate buffered saline  was performed with Zeba Spin Desalting Columns  with 7K MWCO according to the manufacturer’s instructions. For the ddFLN4 construct, the incubation was performed at room temperature. All other steps were done in the same way as for GFP.GFP and ddFLN4 constructs were biotinylated using the ybbR-tag/Sfp-Synthase system . For theGel electrophoresis was performed using Any kD Mini-PROTEAN TGX Precast Protein Gels  in TRIS-based running buffer . For lanes 2–4, we heated 0.6 µM SA dissolved in loading buffer  for 5 minutes to 95°C. For the other SA containing lanes, we used about 1.5 µM. For lanes 10–13, we added 1 µl of the purified Sfp reaction mixture containing both biotinylated and un-biotinylated GFP. We employed Precision Plus Unstained Protein Standards  as molecular weight standards. The gel was run at room temperature with a constant current of 25 mA. The gel was analyzed with a ChemiDoc MP Imaging System .280 = 167,760 M-1cm-1 calculated from the protein sequence using the SIB bioinformatics resource portal [The calorimetric experiments were carried out with a Malvern MicroCal ITC200 . SA samples were equilibrated with PBS using Zeba Spin Desalting Columns  with 40K MWCO following the manufacturer’s instructions. The concentration was determined by spectrophotometry with a NanoDrop 1000  using an extinction coefficient of εe portal . Biotin AFM cantilevers  and glass coverslips were silanized as described by Zimmermann et al. . They weThe experiments were performed with a custom-built AFM as described by Gumpp et al. . The canStreptomyces avidinii (sSA) shows only one slightly larger and broader band. In contrast to the recombinantly expressed core SA monomer that consist of 123 residues, the SA monomer from Streptomyces avidinii contains 183 amino acids. In a posttranslational digest process, it is cut down to core SA.After expression and purification, we checked size and quality of the SAs with SDS polyacrylamide gel electrophoresis . HeatingThe size of the tetramers can be estimated from unheated samples . For mSATo illustrate the binding stoichiometry of the SAs to biotin, we added biotinylated GFP to mSA, tSA, and sSA . Since tKD is lower than 1 nM. The binding enthalpy per mole of added biotin  and the binding stoichiometry  confirmed that the functional subunit of our modified mSA is capable of binding biotin in the same manner as the subunits of sSA, while the binding of biotin to the mutated non-functional subunits is negligible. The measured enthalpies are also in line with previously reported values [We compared the binding properties of our modified mSA with tSA and sSA by isothermal titration calorimetry . Becaused values . This imUsing reconstituted mSA in combination with a calibration domain, we were able to perform SMFS with a well-defined pulling geometry that are not distorted by SA’s multivalency. In our experiments, force propagates only through a single subunit of the SA tetramer . TherefoIn an AFM experiment, about 5,000 force extension traces were recorded of which about 1,100 showed interaction. A larger data set of over 50,000 traces obtained in a 15 h measurement is shown in the supplement . To provFor every data bin along the extension axis, we selected the force bin with the highest value to obtain a characteristic force-extension curve. The curve consists of three parts: First, only the PEG-spacers on the cantilever and the surface are stretched . Then ddFrom the worm-like chain model, an expression for the contour length as a function of persistence length, force and extension can be derived . AssuminWe probed the biotin:mSA complex with five different retraction velocities . The distributions of the resulting forces of the biotin:mSA unbinding and the ddFLN4 unfolding are depicted in Δx0 and the zero-force off-rate koff,0 can be determined. For the ddFLN4-unfolding, we find Δx0 = (0.76 ± 0.05) nm and koff,0 = 8 × 10-4 s-1 for the first unfolding peak and Δx0 = (0.56 ± 0.02) nm and koff,0 = 5 × 10-2 s-1 for the subsequent peak. The distance to the transition state of the biotin:mSA unbinding reads Δx0 = (0.38 ± 0.02) nm and the zero-force off-rate is determined as koff,0 = 3 × 10-6 s-1. The off-rate is in good agreement with the value obtained in an off-rate assay  [The dynamic force spectrum is shown in 0−5 s-1) . Previou0−5 s-1) . For theEven though binding of biotin to SA is widely used as a tool and has been extensively studied previously, the unbinding forces reported in the literature scatter substantially. With the development of mSA and progress in AFM-based SMFS it became possible to study the mechanical stability of the biotin:SA complex in a better defined way. Relating to previous measurements of the unbinding of biotin from tetravalent SA, we illustrated how multivalency of receptor molecules can distort SMFS data of receptor-ligand unbinding. We presented AFM-based SMFS data of the unbinding of biotin from monovalent SA with a 1:1-stoichiometry in a distinct pulling geometry, in which the force only propagated through a single subunit of the SA tetramer. The main improvements of our measurements contributing to the high quality of our data are covalent immobilization of both receptor and ligand molecules, the use of a calibration domain to verify single-molecule interaction events, and exact control over the attachment geometry by a single distinct anchoring site and monovalent receptor molecules.Beyond that, we introduced a new tethering strategy for the use of mSA not only in force spectroscopy but also in many other single-molecule applications. The immobilization of mSA by implementing a single cysteine at the terminus of the functional subunit provides an anchoring site for sulfhydryl-reactive chemical groups, i.e. an anchoring site that is orthogonal to the interaction with biotin. In contrast to defined divalent SA  that canS1 Appendix(PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.S3 Appendix(PDF)Click here for additional data file.S4 Appendix(PDF)Click here for additional data file.S5 Appendix(PDF)Click here for additional data file.S6 Appendix(PDF)Click here for additional data file.S7 Appendix(PDF)Click here for additional data file.S8 Appendix(PDF)Click here for additional data file.


Carbapenem antibiotics are considered the treatment of choice for serious extended-spectrum beta-lactamase (ESBL)-producing Gram-negative bacteria (GNB) infections. The study objectives were to evaluate efficacy and safety of de-escalation therapy to ertapenem for treatment of infections caused by extended-spectrum-β-lactamase-producing Enterobacteriaceae.Enterobacteriaceae infections who had received any group 2 carbapenem for less than 96 h. In the intervention group, the previously-prescribed group 2 carbapenem was de-escalated to ertapenem. In the control group, the group 2 carbapenem was continued.We conducted a randomized controlled trial of adult patients with documented ESBL-producing P = .05), a significantly shorter median length of stay , and a significantly lower defined daily dose of carbapenem use .During June 2011–December 2014, 32 patients were randomized to the de-escalation group and 34 to the control group. Most common sites of infection were urinary tract infection (42%). Characteristics of both groups were comparable. By using a 15% predefined margin, ertapenem was non-inferior to control group regarding the clinical cure rate , the microbiological eradication rate (%Δ = 4.1 [−5.0 to 13.4]), and the superimposed infection rate (%Δ = −16.5 [−38.4 to 5.3]). Patients in the de-escalation group had a significantly lower 28-day mortality rate (9.4% vs. 29.4%; Enterobacteriaceae infections, once the susceptibility profiles are known. Future studies are needed to investigate ertapenem efficacy against ESBL-producing Enterobacteriaceae pneumonia to determine its applicability in life-threatening conditions.Ertapenem could be safely used as de-escalation therapy for ESBL-producing NCT01297842. Registered on 14 February 2011. First patient enrolled on 27 June 2011.ClinicalTrials.gov identifier:  Empirical broad-spectrum antimicrobial therapy is highly recommended in patients with serious infections in aiming to improve patient’s outcomes . HoweverEnterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter spp. As compared to group 2 carbapenems, ertapenem is not active against P. aeruginosa and Acinetobacter spp. . Treatment outcomes are reported as % difference  and 95% CI. Intention to treat analysis was used for all analyses. All statistical calculations were performed using STATA version 14.0/IC .Based on data obtained from our pilot study [unpublished data], the estimated clinical cure rate of group 2 carbapenems in the treatment of ESBL-producing Enterobacteriaceae infection were empirically treated with group 2 carbapenems at our medical center. Sixty-six of those patients satisfied the inclusion criteria and were enrolled in this study . Approximately 40% of patients in both groups had urinary tract as the site of ESBL-producing Enterobacteriaceae infection. Fourteen patients in the de-escalation group (43.8%) and 12 patients in the non-de-escalation group (35.3%) had primary bacteremia. The mean APACHE II score and mean modified APACHE II score were comparable between groups. Mean time from onset of infection to enrollment was 4.2 ± 1.6 days in the de-escalation group and 4.5 ± 1.2 days in the non-de-escalation group (P = .47). There was no significant difference in duration of pre-enrollment use of group 2 carbapenems  or duration of pre-enrollment use of any other antibiotic  between the de-escalation and non-de-escalation groups, respectively.Twelve patients (37.5%) in the de-escalation group and 20 patients (58.8%) in the non-de-escalation group had a healthcare-associated infection  and in the non-de-escalation group (control) are shown in Table p = 0.13). The three leading causative pathogens of superimposed infection were MDR A. baumannii, methicillin-resistant Staphylococcus aureus, and Candida spp. There was no significant difference in the distribution of causative pathogens between groups. Patients in the de-escalation group had a significantly shorter median length of hospital stay  and a significantly lower defined daily dose (DDD) of carbapenem antibiotic use . There were no significant differences in stool colonization by MDR pathogens at enrollment or at the end of therapy.Superimposed infection rate was slightly lower among patients in the de-escalation group . There was no statistical difference in ADRs between groups.The findings of this study indicate that de-escalation therapy to ertapenem is non-inferior to continuation of group 2 carbapenems for clinical cure rate, microbiological eradication rate, and superimposed infection rate. These findings confirm the efficacy of ertapenem and are consistent with the findings of previously documented in many observational cohort studies –14. The Similar to results from a recent systematic review, our study did not find any significant difference in stool colonization by MDR pathogens. However, our baseline rate of colonization with resistant GNB pathogen was relatively high (>60%), compared with the studies included in another systematic review .Patients in the de-escalation group also had a significantly shorter length of hospital stay, as well as a significantly lower defined daily dose (DDD) of carbapenem use. This can be simply explained by the practice of once-daily dosing of ertapenem , wherebyThis study has several inherent strengths. While most previous studies were observational, this was a randomized controlled trial. Randomization ensured a balance of measured and unmeasured confounders between the intervention and control groups. In addition to the evaluation of clinical outcomes, other important parameters, including stool colonization with resistant GNB and cost-related outcomes were evaluated.Enterobacteriaceae pneumonia. Third, the study was early terminated before achieving the target sample size, therefore all statistically significant findings are less reliable. Lastly, the study was designed to stratify only on the site of infection but did not stratify on other clinically significant factors such as age and APACHE score. Nevertheless, these two important factors were comparable between two groups.This study also has some mentionable limitations. First, participation bias and observer bias may have occurred because the study design was open-label. However, the microbiological cure rate and 28-day mortality rate are objective measures, so observer bias would have been minimal. Second, the majority of patients in this study had UTI or primary bacteremia, with only a small proportion having pneumonia. Thus, we are not able to conclude that de-escalation to ertapenem is effective against ESBL-producing Enterobacteriaceae infections, especially UTIs and primary bacteremia. Future studies are needed to investigate ertapenem efficacy against ESBL-producing Enterobacteriaceae pneumonia to determine its applicability in life-threatening conditions. Additionally, a study in the cost-effectiveness of de-escalation to ertapenem would be useful for healthcare policymakers.Based on the results of this study, ertapenem can be safely used as de-escalation therapy for ESBL-producing


The VascuQoL-6 (VQ-6) health-related quality of life questionnaire, a short version of the disease-specific VascuQoL-25, was developed for clinical practice and use in vascular registries. The study purpose was to evaluate the validity and reliability of VQ-6.VQ-6 was translated to Norwegian with linguistic validation and face value evaluation, and consecutive patients with intermittent claudication (IC) or critical limb ischemia (CLI) were included. All patients completed VQ-6 and Short Form-36 (SF-36), and were evaluated with ankle-brachial index (ABI) measurement pre- and post-exercise, a constant load treadmill test and clinical consultation at baseline and after 4 weeks. Correlation analysis, change statistics and receiver operator characteristics (ROC) curves were used to evaluate reliability, validity and responsiveness to change.r = 0.55–0.62) and excellent responsiveness to change after treatment, standard response mean (SRM) 1.12. The clinical anchors of ABI at rest, treadmill walking performance and Fontaine class improvement were less responsive to change than VQ-6, SF-36 and the vascular surgeon’s evaluation.One hundred seventy-one patients with peripheral arterial disease (PAD) were included, 70 (41%) female. 147 (86%) of the patients suffered from IC. The reliability of VQ-6 was good, Cronbachs-α 0.82. The ability of VQ-6 to differentiate between IC and CLI was good, area under the curve (AUC) 0.754. There was good correlation between SF-36 physical domains and component scores and VQ-6 score . Patient reported outcome measures (PROM) and patient reported experience measures (PREM) are increasingly important in the evaluation of health care quality. In quality registries, such measures can add important information to the outcome evaluation.PROMs are usually based on questionnaires . For patThe symptoms of PAD vary from leg claudication (intermittent claudication –IC) to pain at rest and gangrene , and the natural history of the disease spans from stable disease to the need for vascular reconstruction or leg amputation. In vascular registries, treatment outcome for CLI can be measured through patency of vascularized vessel segments, limb salvage and amputation-free survival. However, the evaluation of outcome after revascularization in low risk patients suffering from IC is more challenging. The severity of claudication symptoms and restrictions in daily life are important factors in determining whether the patient should be offered invasive treatment . To inclThe Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II)  recommenhttp://www.ucr.uu.se/swedvasc/), but VQ-6 has not been validated in a separate study.The VascuQoL is a questionnaire developed in the UK for research purposes in 2001 . The oriThe process of validation for a health-related QoL measure is not a simple or finite task, but requires a continuum of evidence based on a series of investigations to assess meaning and usefulness . ValidatThe aim of this study was to evaluate the validity, reliability and responsiveness of VQ-6, a disease-specific QoL measurement for use in clinical practice and vascular registries.Consecutive patients with new referral for evaluation of peripheral arterial disease (IC or CLI) at the vascular surgery department at two different hospitals (H1 and H2) were invited to participate based on the information given by the referring physician. The inclusion period ran from August 2014 to August 2015. Patients received written information about the study and the two questionnaires, VQ-6 and SF-36, by post before the scheduled appointment at the outpatient clinic. If the patients failed to bring the questionnaires or were admitted acutely, they were invited to participate at site and completed the questionnaires before further investigations. The returned questionnaires were not available to the treating physician.If the consulting vascular surgeon ruled out symptomatic PAD, the patients were excluded. For the patients who died, underwent major amputation or major non-vascular surgery, or moved out of the hospital region, only baseline data was available. Patients also had the opportunity to withdraw at all times. Inclusion, exclusion and follow-up of patients are shown in Fig. In the vascular laboratory, arterial pressures were measured with a hand held Doppler device (H1: Model 811-BTS (9.5 MHz) Parks medical electronics INC, Aloha, Oregon, USA, H2: Model 811-BTS (8.2 MHz) Parks medical electronics INC, Aloha, Oregon, USA), and the ankle-brachial index (ABI) was calculated. Patients with claudication were tested on a constant load treadmill with a speed of 2.5 km/h and no inclination . A post-exercise ABI drop exceeding 0.1 was regarded significant. Intermittent claudication distance (ICD) and maximum walking distance (MWD) were registered in meters. There is a ceiling effect at 416 m, as the test was terminated after 10 min for all patients.Risk factors, comorbidity, medication and Fontaine classification  were regAll patients were scheduled for a new consultation with completion of questionnaires, arterial pressure measurements, treadmill-test and clinical evaluation. Patients receiving conservative treatment  and patients referred for supervised exercise therapy were followed up after 4 weeks. The referral algorithm for imaging and invasive treatment was unaltered from usual practice. If results from imaging indicated a conservative approach, the patients had their follow-up as soon as possible. Patients referred for invasive treatment  underwent follow-up 4 weeks after the invasive procedure.The VQ-6 was translated to Norwegian from the Swedish version using the method of linguistic adaption and validation described by the MAPI institute , includiEach of the six items scores from one to four, sum score range is from six to 24, and a higher score indicates better health.SF-36 (version 1) was chosen as generic QoL anchor, as this questionnaire has been used in prior validation of VascuQoL-25 , 13, 17 The subscale scores of SF-36 range from 0 to 100, and the highest score indicates no health-related reduction of QoL. The component summary scores relates to the normative population , and a score lower than 50 indicates lower QoL than the normative population.www.norkar.no) showed an approx. 15–18% proportion of CLI at H1. Earlier studies have shown 20–32% improvement in VascuQoL-25 summary score after invasive treatment [The power analysis was conducted for the responsivity to change after treatment. An evaluation of the practice at H1 showed that one third of patients referred with IC and most patients with CLI would receive invasive treatment. Registrations of invasive treatment from the Norwegian vascular registry .Cronbach’s alpha was calculated to evaluate the internal consistency of the VQ-6. A reliable health-related QoL-measure should center between 0.7 and 0.9 for group comparisons . The intContent validity was assessed through face value evaluation and through the earlier development processes , 12.The construct validity  was assessed through the instrument’s ability to discriminate between patients suffering from IC and CLI, using Receiver operating characteristic (ROC) curve analysis, with the Fontaine classification as a basis. The area under the ROC curve (AUC) benchmarks were > 0.8 (excellent), 0.7–0.8 (fair) and < 0.7 (poor).Criterion validation was performed through exploration of correlations between SF-36 domain and summary scores and VQ-6 sum scores, using Pearson product-moment correlation coefficient for normally distributed variables and Spearman’s rank correlation coefficient as a non-parametric alternative. Interpretation of correlation coefficients was done using Cohen’s criteria: small 0.1–0.29, medium 0.3–0.49 and large 0.5–1.0.Standard and hierarchical linear multiple regressions were used to model the relationship between the questionnaires while controlling for other variables, after checking for violation of assumptions .Change analysis was done using t-test for normally distributed continuous data, Wilcoxon signed rank test for non-parametric continuous data and McNemar’s test for dichotomous data.ROC curves were calculated for comparison with clinical anchors of change.Standardized response means (mean change divided by the standard deviation of the change) of SF-36 domains and component summary scores and all items and summary score of VQ-6 were calculated to evaluate responsiveness .To determine the clinically important change in VQ-6, the minimally important difference (MID) was calculated using the distribution based method with 0.5 SD  as well Analysis was performed using all available data at baseline and during follow-up. If only one item was missing for VQ-6 (3 patients) we did a single imputation using the median of the item score for all patients. One patient missed one item after treatment, and the sum score of VQ6 was omitted from analysis. For SF-36, imputation was done using the scoring software . The mean subscale score is used if the patient has answered more than half of the items in the domain.One hundred seventy-one patients were included, 41% female. Some 31% had a previous history of evaluation or treatment for PAD. Patient characteristics are given in Table n = 171), the VQ-6 demonstrated good internal consistency .As determined by Cronbach’s alpha coefficient calculations at baseline , 19 patients (28%) improved four points or more in VQ-6 summary score after 4 weeks. There was a statistically significant improvement in VQ-6 summary score and SF-36 PCS mean. The SF-36 MCS, ABI at rest, ABI post-exercise and MWD was unchanged from baseline . In the final model, only the contribution of PF and BP were statistically significant .Hierarchical multiple linear regression was used to assess the ability of the generic health related QoL measure (SF-36), ABI at rest and the patients’ ability to participate in a treadmill test to predict the disease-specific QoL (VQ-6 summary score) at baseline, after controlling for age, gender, BMI, smoking status and comorbidity. The total variance explained by the model was 59%. The factors controlled for could explain 4.7% of the variance in VQ-6 summary score. The SF-36 domains, ABI at rest and ability to perform on a tread-mill explained 54.1% of the variance after controlling for age, gender, BMI, smoking and comorbidity . This hierarchical regression model included the SF-36, ABI at rest, an ABI drop > 0.1 and MWD, controlling for age, gender, BMI, smoking and comorbidity. The total variance explained by the model was 58.2%. The factors controlled for could explain 3.7% of the variance in VQ-6 summary score. The SF-36, ABI at rest, ABI drop and MWD explained 54.5% of the variance after controlling for age, gender, BMI, smoking and comorbidity .A separate regression model was used to assess only the patients able to perform the walking test , 41 patients (56%) improved four points or more in VQ-6 summary score. There was a statistically significant improvement in VQ-6 summary score and all domains except RE for the invasive group, compared to only two domains for the conservative group (PF and BP).In the invasive group . To evaluate reliability through test and retest, ICC was calculated for the conservative group, and the result of 0.66 indicates good reliability, but may be underestimated in this study, the observed improvement in QoL scores for the conservative treatment group taken in account. As the conservative group was restricted to claudicants, reliability has not been tested with ICC for patients with CLI.SF-36 is a profile, while VQ-6 gives an index. This complicates the comparison of these instruments. The component summary scores of SF-36 are less sensitive to changes affecting only a few domains, as for patients with PAD. Low scores in physical domains will inflate the MCS and visa versa .Since the two instruments were administered together, external factors not specific to the disease  could influence the answers to both questionnaires, and lead to a higher correlation than the underlying traits account for . This efVascuQoL is intended to cover the spectrum of severity of PAD. Patients with CLI will probably have a lower score due to pain and concern, while patients with IC have more restrictions in activity. As the short form (VQ-6) contains two items about activity , the relationship between VQ-6 summary score and clinical improvement may be greater for patients with IC.Clinically significant improvement or deterioration needs to be translated into points of change in results from the questionnaire in the individual patient. The MID indicates the lowest change in score that can be interpreted as improvement or deterioration relevant to the patient. For VQ-25 the MID has been discussed in recent articles by Conijn , 34. As VQ-6 is a reliable and valid instrument for evaluation of QoL in patients suffering from PAD in clinical practice, and the summary score can be used in group comparisons, for instance in vascular registries. PROMs constitute an important individual outcome measure not covered by physical measurements of ABI and walking capacity.


Vibrio alginolyticus infection, confirming its participation in the innate immune system of shrimp. The anti-miRNA oligonucleotide (AMO-miR-100) was applied to inhibit the expression of miR-100. After AMO-miR-100 treatment, the shrimp was challenged with WSSV or V. alginolyticus. The knockdown of miR-100 expression decreased the mortality of WSSV-infected shrimp from 24 h to 72 h post-infection and enhanced the mortality of V. alginolyticus-infected shrimp significantly. The knockdown of miR-100 affected phenoloxidase (PO) activity, superoxide dismutase (SOD) activity and total hemocyte count (THC) after the infection with WSSV or V. alginolyticus, indicating a regulative role of miR-100 in the immune potential of shrimp in the response to WSSV or V. alginolyticus infection. The knockdown of miR-100 induced the apoptosis of shrimp hemocytes, and V. alginolyticus + AMO-miR-100 treatment caused more hemocyte apoptosis than V. alginolyticus treatment. The miR-100 influenced also the morphology of shrimp hemocytes and regulated the phagocytosis of WSSV or V. alginolyticus. Thus, we concluded that miR-100 may promote the anti-Vibrio immune response of shrimp through regulating apoptosis, phagocytosis and PO activity and affects the progression of WSSV infection at a certain level.In this study, we discovered that shrimp miR-100 was up-regulated at 24 h after WSSV or  V. alginolyticus infectionMicroRNAs (miRNAs) are small non-coding RNAs that function by base pairing with messenger RNAs and are involved in the regulation of almost every biological pathway1235789101113Mus musculus, Danio rerio, Homo sapiens, Xenopus tropicalis, Apis mellifera, and Bombyx mori. MicroRNA-100 (miR-100) has been confirmed to act as a tumor suppressor in various types of human cancer, including epithelial ovarian cancer, non-small cell lung cancer, colon cancer, hepatocellular carcinoma, glioblastoma, osteosarcoma, and colorectal cancer161718192021Vibrio alginolyticus  infection in shrimpVibrio sp. can infect not only invertebrates such as shrimps and crabs, but also vertebrates, including fishes and mammals. Vibrio sp. prefers hypersaline habitats, which makes Kuruma shrimp (Marsupenaeus japonicus), an important to humans species abundantly present in the ocean, the main victim of one of its main victims. WSSV is another lethal pathogen of Kuruma shrimps, and the infection caused by WSSV can lead to over 90% mortality in one week. In this study, we further explored the role of miR-100 in the innate immunity response of shrimp to WSSV or Vibrio infection.MicroRNA-100 is exceedingly conservative, and its sequence is identical in model species, such as V. alginolyticusof shrimp miRNAs response to WSSV or infection. Our results indicated that the expression of a number of miRNAs was significantly up-regulated 24 h after WSSV infection and down-regulated 24 h following V. alginolyticus infection . We choose these immune-related genes for their known function. Target genes and pathways of miRNA-100 were predicted by TargetScan and miRanda algorithms. The target gene of miRNA-100 is importin subunit alpha-3 and the pathway is protein transporter activity. This result makes no mean to our research, because we want to investigate the function of miRNA-100 only. The data revealed that hemocyanin was significantly (P < 0.01) up-regulated by the inhibition of miR-100, whereas myosin was significantly (P < 0.01) down-regulated  . The resegulated .V. alginolyticus-infected shrimp group, the shrimp group treated with AMO-miR-100 was challenged with V. alginolyticus. The results revealed that the AMO-miR-100 treatment significantly (P < 0.01) elevated the mortality of V. alginolyticus-infected shrimp when compared to that of the group with V. alginolyticus challenge only  lower than that of the WSSV group 36 and 48 h post-treatment. This result showed that the knockdown of miR-100 weakened the PO activity after the WSSV infection, indicating that miR-100 can regulate the proPO system of shrimp in response to WSSV infection.After WSSV infection, the PO activity was clearly increased and over 10% higher than that in the PBS control . It is nV. alginolyticus, the PO activity in the infected group was slightly higher than that in the PBS control, but no significant fluctuation was observed as infection time increased  than that of the V. alginolyticus group at 12 h post-treatment. Within the interval from 24 h to 48 h post-treatment, the PO activity of the V. alginolyticus + AMO-miR-100 group deceased even further in comparison to that of the V. alginolyticus group. This result suggested that the knockdown of miR-100 affected PO activity after the infection with V. alginolyticus, indicating a regulative role of miR-100 in the proPO system of shrimp in response to V. alginolyticus infection.After the infection with ncreased . The PO Vibrio. alginolyticus infection, the SOD activity of shrimp hemolymph dropped below control at 12 h, then raise over 1.5 times higher than PBS group 24–36 h, declined again at 48 h  than that of the V. alginolyticus + AMO-miR-100 treatment . In the PBS treatment, the percentage of Annexin V-positive hemocytes was 29.0% , which wreatment . This reV. alginolyticus + AMO-miR-100 treatment (18.1%) was significantly higher (P < 0.01) than that in the V. alginolyticus group (9.5%). These data suggest that the knockdown of miR-100 would lead to more pronounced V. alginolyticus engulfing by phagocytes. This result indicates that miR-100 may negatively regulate the phagocytosis progress.To observe the cell morphology of hemocytes, they were stained and analyzed under a confocal microscope, and then the phagocytosis was detected by flow cytometry. The results showed that, in comparison with the control group, the cell extension of AMO-miR-100-treated hemocytes was not good as that of the control, and the cell morphology was abnormal, especially concerning the cytoskeleton . TherefoV. alginolyticusV. alginolyticus infections.Increasing evidence indicates that miRNAs participate in almost every step of cellular processes and are often aberrantly expressed in human cancer34516171819202123In loss function experiment, we found there were two genes, among multiple major immune related genes, were influenced by the knock-down of miR-100. Hemocyanin, and Myosin. Previous research has shown that hemocyanin is an important respiratory protein which transports oxygen throughout the bodies of shrimps27282931V. alginolyticus infection would not.Recently, many studies have confirmed that miR-100 can regulate multiple genes to induce the cell apoptosis in many types of mammalian cell lines. There is a growing body of evidence that miR-100 inhibits the progression of apoptosis in human cancer cells through multiple pathways. Shrimp miR-100 was found to be an inhibitor in the apoptosis process of shrimp hemocytes in previous reportV. alginolyticus infected shrimps, the PO activity was slightly increased at early stage, and SOD activity was relatively lower and showed a less activity increasing trend. The less effective function of PO and SOD implying a positive role of miR-100 in the anti-virus and anti-Vibrio respond. The THC post WSSV infection increased under miR-100 knock-down, while the THC post V. alginolyticus infection decreased. The same results were obtained in the apoptosis analyze. The mortality of V. alginolyticus-infected shrimps was also elevated. Meanwhile, the knockdown of miR-100 resulted in an increase in the phagocytosis rates of both WSSV-infected and V. alginolyticus-infected shrimps. This result indicated that miR-100 also inhibited the phagocytosis ability of shrimp hemocytes.To explore other effect of miR-100 on shrimp innate immunity, several functional parameters for evaluating the immune potential have been detected. In WSSV-infected shrimps, as the miR-100 expression was inhibited, the PO activity and SOD activity was significantly decreased. In Vibrio immune response of shrimp through regulating THC, apoptosis, phagocytosis, and PO activity. Furthermore, it exerts an influence on WSSV infection at a certain level via controlling SOD activity, hemocyte phagocytosis and PO activity.Synthesized into a conclusion, these findings indicate that shrimp miR-100 plays an essential role in the innate immune system of shrimp. miR-100 promotes the anti-M. japonicus individuals were obtained from the seafood market of Hangzhou, Zhejiang, China. The hemocytes were collected from infected and uninfected shrimps. The samples were immediately used for RNA extraction to prevent total RNA degradationTotal of healthy adult V. alginolyticus was cultured and used to challenge the shrimps accord to a protocol reported earlierWSSV (GenBank accession no. AF 332093.1) was purified and used in a challenge experiment as previously describedTotal RNAs were extracted from the hemocytes of the infected and uninfected shrimps at 24 h and 48 h post-infection by using a miRNA isolation kit  in accordance with the manufacturer’s protocol. The next steps were performed following the guidelines outlined in a previously mentioned reportThe small RNAs were extracted from shrimp hemocytes using mirVana miRNA isolation kit  according to the manufacturer’s manual. The concentration of the small RNA was measured using NanoDrop, and 3 mg small RNAs per sample were used for Northern blotting as previously describedTo silence the expression of miR-100(AACCCGTAGATCCGAACTTGTG), the anti-miRNA-100 oligonucleotide (AMO-miR-100) was injected into each shrimp at a dose of 0.15 mM/shrimp. As a control, the sequence of AMO-miR-100 was scrambled, and shown as AMO-miR-100-scramble. The AMO-miR-100-scramble (0.15 mM/shrimp) and phosphate buffered saline (PBS) were included in the injections. To avoid the degradation of AMO-miR-100 or AMO-miR-100-scramble, the oligonucleotides were sequentially injected into the same shrimp three times within a 24-h interval. Twenty-four hours after the last injection, the shrimp hemocytes from three random shrimp individuals were mixed and subjected to Northern bolt and phagocytosis assays. All the assays described above were biologically repeated three times.Total RNA of hemocytes was extracted by Easy spin tissue/cell RNA extra kit  following the protocol of manufactures. Experiments were performed in triplicates, and at least three shrimps were analyzed for each type of tissue. Less than 200 μg total RNA was used for cDNA synthesis by ReverTra Ace qPCR RT Master Mix with gDNA Remover Code: FSQ-301 . The cDNA was stored at −20 °C. A SYBR Green qRT-PCR assay was carried out in a Bio-Rad Two Color Real-Time PCR Detection System, and the data were calculated accord to the 2−ΔΔCT comparative CT method using Microsoft Office Excel 2007, with the amplification of GAPDH as an internal control. Designing and synthesizing of the qRT-PCR primers were entrusted to Generay Shanghai Company, based on the open read frame (ORF). The primer sequences for SYBR Green RT-PCR are shown in mirVanaTM miRNA Isolation Kit  following the protocol of manufactures. Experiments were performed in triplicates, and at least three shrimps were analyzed for each type of tissue. Less than 200 μg total RNA was used for cDNA synthesis by TaqManTM MicroRNA Reverse Transcription Kit . The reverse transcription primer of miR-100 and real-time PCR primers of miR-100 and U6 were designed based on Stem-Loop miRNA primer design principle, the sequences were showed in Total RNA of hemocytes was extracted by 5 WSSV copies/mL) containing AMO-miR-100 by using a syringe with a 29-gauge needle. As controls, shrimps  were injected with PBS only or WSSV only. After the last injection, the mortality of shrimps was monitored every day.Shrimp  were injected intramuscularly with AMO-miR-100 in the lateral area of the fourth abdominal segment. Twelve hours later, the treated shrimps were injected with 0.1 mL of filtrate 6. The separate cells were divided into three equal tubes used as a control, WSSV, and VA group, respectively. FITC-labeled WSSV or VA was added in the hemolymph cells accord to the procedure described in a previous reportAfter purchase, the shrimps were stored in ice-cold sea water until use. Then, the shrimps were sterilized with 70% alcohol, and hemolymph was withdrawn and mixed 1:1 in a disposable syringe containing 20 mM EDTA. Next, to collect hemolymph cells, the samples were centrifuged at 2000 rpm x 10 min at 4 °C. The cells were gently suspended thoroughly with high salty PBS under aseptic conditions, the cell count number was determined by an auto cell counter, and their density was adjusted to 3–5 × 10V. alginolyticus, respectively, while the other two groups were treated with a mixture of AMO-miR-100 and pathogens  of each group were incubated with 10 uL of L-DOPA in 80 µL saline for 15 min at room temperature. The incubated serums were applied to measure the PO activity using a BioRad spectrophotometer with detection at a wavelength of 490 nm.To determine the influence of miR-100 on shrimp PO activity, healthy shrimps were divided into five groups: three groups were treated with PBS, WSSV, and Shrimp hemolymph of each group was extracted for SOD extraction. 100ul hemolymph was homogeneous mixed with 0.5 ml phosphate buffer , then centrifuged at 5724 × g for 5 min, 4 °C. The supernatant was separated into a clean new tube, followed by 65 °C heating for 5 min, then centrifuge for 5 min. Separated the new supernatant, and store at −20 °C for further assay. To avoid protein degeneration, samples were kept on ice always.SOD activity was determined using Nitro blue tetrazolium method (NBT) created by BeauchampThe specific SOD activity unit was defined as the enzyme amount that can induce half reaction rate of the initial reaction rate (without enzyme). The formula below was applied to calculate SOD activity units. SOD unit of PBS groups were used as an index to compare the different effect of treatments on the SOD activity. The results were expressed as Relative SOD activity.SOD activity unit = (OD560control-OD560sample)/50% OD560control * dilution ratioApoptosis assay of shrimps with/without miR-100 knockdown was conducted with Annexin V  accord to the manufacturer’s protocol. Shrimp hemolymph from the treatment groups were harvested in 1:1 with 20 mM EDTA, centrifuged at 2000 rpm x 10 min to collect hemocytes, and washed once in pre-cold high-salt PBS. The hemocytes were gently suspended in 1 × Annexin-Binding buffer. Then, 5 μL of Alexa Flour 488 Annexin V and 1 μg/mL of PI (propidium iodide) were added. The samples were incubated at room temperature for 15 min or at 4 °C for 30 min. Next, to end the reaction, 400 μL of 1 × Annexin-binding buffer was added into the samples. Finally, the fluorescence emission of the stained samples was examined by flow cytometry at wavelengths of 530 nm and 575 nm.t-test.The data from three independent experiments were analyzed by one-way analysis of variance (ANOVA) to calculate the mean and standard deviation of the triplicate assays. The differences between the different treatments were analyzed by How to cite this article: Zhi, W. and Fei, Z. MicroRNA-100 is involved in shrimp immune response to white spot syndrome virus (WSSV) and Vibrio alginolyticus infection. Sci. Rep.7, 42334; doi: 10.1038/srep42334 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Nonspecific ST-segment and T-wave (ST-T) changes represent one of the most prevalent electrocardiographic abnormalities in hypertensive patients. However, a limited number of studies have investigated the association between nonspecific ST-T changes and unsatisfactory blood pressure (BP) control in adults with hypertension.The study population comprised 15,038 hypertensive patients, who were selected from 20,702 participants in the China Stroke Primary Prevention Trial. The subjects were examined with electrocardiogram test at the initial visit in order to monitor baseline heart activity. According to the results of the electrocardiogram (defined by Minnesota coding), the subjects were divided into 2 groups: ST-T abnormal and ST-T normal. Unsatisfactory BP control was defined as systolic BP ≥140 mm Hg or diastolic BP ≥90 mm Hg following antihypertensive treatment during the 4.5-year follow-up period. Multivariate analysis was used to analyze the association between nonspecific ST-T abnormalities and unsatisfactory BP control.P = 0.005]), independent of traditional risk factors, as demonstrated by multivariate regression analysis. Notable differences were further observed in male subjects  and in patients with comorbid diabetes .Nonspecific ST-T changes were common in hypertensive adults (approximately 8.5% in the study), and more prevalent in women (10.3%) and diabetic patients (13.9%). The unsatisfactory BP control rate was high in the total population (47.0%), notably in the ST-T abnormal group (55.5%). The nonspecific ST-T abnormal group exhibited a significantly greater rate of unsatisfactory BP control (odds ratio [OR] 1.20, 95% confidence interval [CI] , Greater rates of unsatisfactory BP control in hypertensive patients with electrocardiographic nonspecific ST-T abnormalities were observed, notably in the subcategories of the male subjects and the diabetic patients. The outline of the Report on Cardiovascular Disease in China (2014) demonstrated that the hypertension control (BP <140/90 mm Hg) rate was approximately 9.3% of all patients, and/or 27.4% of treated patients, although 42.6% of hypertensive patients in China were aware of their condition and 34.1% were receiving treatment. Furthermore, the BP control rate was lower in female subjects and rural patients compared with that in male subjects and urban patients, respectively. The electrocardiogram (ECG) is a routine, accessible, cost-effective, and recommended diagnostic tool for the initial evaluation and follow-up of hypertensive patients. In the standard surface ECG, nonspecific ST-segment and T-wave (ST-T) changes are a common finding. Recently, a majority of studies have indicated that nonspecific ST-T abnormalities are significantly associated with cardiovascular morbidity and mortality.–9 However, the prognostic significance of nonspecific ST-T abnormalities for BP control has not been fully investigated.Hypertension is a common chronic disease that affects a great proportion of the population worldwide. It is considered one of the major risk factors for the development of cardiovascular diseases such as stroke, ischemic heart disease, and heart failure.The goal of the present study was to determine whether nonspecific baseline ST-T ECG abnormalities are associated with unsatisfactory BP control in hypertensive patients. The study aimed to provide information on the potential improvement of the management of hypertension.22.1 The detailed description of the stage designed and the methodology have been previously described. Eligible participants were men and women 45 to 75 years of age who had hypertension. The major exclusion criteria included the history of stroke, myocardial infarction, heart failure, coronary revascularization, and/or congenital heart disease. Individuals with a missing ECG measurement, an illegible ECG, and/or ECG abnormalities inconsistent with nonspecific ST-T changes  at baseline .ine Fig.  were furThe CSPPT protocol was approved by the ethics committee of the Institute of Biomedicine, Anhui Medical University, Hefei, China (FWA assurance number FWA00001263). All participants provided written informed consent for their participation in the study protocol. The study protocol was conducted according to the principles of the Declaration of Helsinki that ensure that the safety and well-being of the patients is protected and that the integrity of the data is preserved.2.2The CSPPT was a multicommunity, randomized, double-blinded clinical trial conducted between May 19, 2008, and August 24, 2013, in 32 communities in the Jiangsu and Anhui provinces of China. The present study was a prospective cohort study. The 15,038 participants with baseline ECG measures were stratified according to the presence and/or absence of nonspecific ST-T abnormalities using the Minnesota coding criteria. Eligible participants were subsequently divided into 2 groups: ST-T abnormal and ST-T normal. All ECGs were recorded and analyzed by 2 trained medical professionals. In the case of a disagreement regarding the interpretation of an ECG, a third reading was conducted jointly until a final interpretation of the ECG was achieved.2.32 to V6 (MC 4-3); ST-J depression ≥1.0 mm and ST-segment upward sloping or U-shaped, in any of leads I, II, aVL, or V1 to V6 (MC 4-4); T-wave amplitude zero (flat), negative, or diphasic (negative–positive type only) with <1.0-mm negative phase in leads I, II, V3 to V6, aVL when R-wave amplitude is ≥5.0 mm (MC 5-3); and T-wave amplitude positive and T- to R-wave amplitude ratio of <1:20 in any of leads I, II, aVL, or V3 to V6 when R-wave amplitude in the corresponding leads was ≥10.0 mm (MC 5-4).The Minnesota code (MC) criteria for nonspecific ST-T abnormalities were used, as described by the MC ECG classifications 4-3, 4-4, 5-3, and 5-4. The criteria were defined as follows: no ST-J depression ≥0.5 mm but ST-segment downward sloping and ST-segment or T-wave nadir at least 0.5 mm below the P-R baseline, in any of leads I, II, aVL, or VHypertension was defined as seated resting systolic blood pressure (SBP) of ≥140 mm Hg or diastolic blood pressure (DBP) of ≥90 mm Hg at both the screening and recruitment visits and/or use of antihypertensive medication. The BP of the participants was measured during the follow-up period every 3 months from 2008 to 2013. During each visit, the participants were required to rest in a seated position for at least 5 minutes prior to the measurement. BP was measured 3 times at 5-minute intervals by a trained physician with an electronic sphygmomanometer . The mean of the 3 readings was calculated for the measurement of the BP. The average of 20 measurements at 20 different visits was used as the final BP value. Overall unsatisfactory BP control was defined as SBP of ≥140 mm Hg or DBP of ≥90 mm Hg following antihypertensive treatment.Diabetes mellitus was defined as self-reported clinically diagnosed diabetes or use of hypoglycemic agents or a fasting blood glucose concentration of ≥7.0 mmol/L (≥7.0 mmol/L). Body mass index (BMI) was calculated as weight (in kilograms) divided by height (in square meters).12 at both the baseline and the exit visits were measured by a commercial laboratory kit using a chemiluminescent immunoassay . Serum homocysteine, fasting lipids, and glucose levels at the baseline and the exit visit were measured using automatic clinical analyzers  at the core laboratory of the National Clinical Research Center for Kidney Disease, in Nanfang Hospital, Guangzhou, China.Serum folate and vitamin B2.42 tests for categorical variables and t tests and/or Wilcoxon rank-sum tests for continuous variables, as appropriate. The prospective association between nonspecific ST-T changes and unsatisfactory BP control was examined using Cox proportional hazard regression models following adjustment for the covariates. Multiple linear regression was used to assess the association between ST-T abnormality and change of blood pressure under treatment. Furthermore, multivariate logistic regression analysis was used to evaluate the impact of electrocardiographic nonspecific ST-T abnormalities on unsatisfactory BP control in the subgroup analyses. A 2-tailed P-value <0.05 was considered statistically significant.Data were analyzed using the Empower Stats software. The results are presented as mean ± standard deviation for continuous variables and frequence(percentage) for categorical variables. Baseline characteristics were compared between participants in the presence and/or absence of nonspecific ST-T abnormalities using χ3The study population included 15,038 participants (62.1% women) from the CSPPT, which is representative of a low- to medium-risk Chinese hypertensive population. The BP control rate for the total population was 53.0%. The prevalence of nonspecific ST-T abnormalities in the population investigated was 8.5%, whereas prevalence was greater in women (10.3%) and diabetics (13.9%).The baseline characteristics are shown in Table 12, total cholesterol level, high-density lipoprotein cholesterol, and BMI (Table P = 0.005]), whereas with regard to the stratified categories significantly different results were obtained in male  and not in female subjects .The presence and absence of nonspecific ST-T abnormalities significantly differed among related demographic and clinical variables Table . This trP < 0.001, and β 0.50, 95% CI , P = 0.002, respectively). In addition, these patients revealed lower values in the relative percentage decrease in SBP and DBP . Importantly, the aforementioned differences were noted in male and not female subjects.The associations between baseline electrocardiographic ST-T abnormalities and changes in BP following treatment are shown in Table P = 0.002) and subjects with comorbid diabetes .Table 4The major advantage of the present study was the large sample size derived from the CSPPT. The CSPPT was a large randomized trial conducted in adult subjects with hypertension in China without a history of stroke or myocardial infarction. The results of the trial demonstrated that enalapril–folic acid therapy significantly reduced the relative risk of an initial stroke incident by 21% compared with enalapril monotherapy. Given the well-characterized population, the standardized assessment of ECGs using the MC criteria, and the 4.5-year longitudinal follow-up period, the CSPPT study provided a unique opportunity to assess the prevalence of nonspecific ST-T changes in order to identify differences between genders and to explore whether nonspecific ST-T changes were associated with increased risk of unsatisfactory BP control. The study further provided insight regarding the potential predictive value of unsatisfactory BP control. The possible explanations of the aforementioned findings may be the rigorous design of the CSPPT and the strict follow-up schedule applied by trained research staff and physicians, which ensured high adherence of patients to the prescribed treatment regimen. The control rate of the present study conducted in China was similar to that noted in 2 studies conducted in America  and Italy .In the present study, the rate for the achievement of target BP levels of the total population was 53.0% and that was considerably greater compared with that previously reported in a national survey among adult participants in China (27.4% among treated hypertensive participants).,12–16 The greater prevalence of nonspecific ST-T changes in women has been attributed to the interplay of a variety of factors of anatomic, structural, hormonal, autonomic, and genetic origin. High blood glucose is associated with comorbidities such as hypertension, hyperlipidemia, and a prothrombotic state that interact synergistically to promote cardiac changes. The latter changes in turn result in ECG abnormalities, including nonspecific ST-T changes.The current study demonstrated that nonspecific ST-T changes in the ECG of hypertensive patients can be used for the identification of a greater risk of unsatisfactory BP control, notably in male participants and patients with diabetes mellitus. The prevalence of nonspecific ST-T abnormalities was 8.5%, and the prevalence was greater in women (10.3%) and in patients with diabetes (13.9%). This finding is consistent with previous studies.,18 As a result, nonspecific ST-T changes could be considered an early indicator of poor BP management. Following adjustment for the covariables, the current study indicated that hypertensive patients with baseline nonspecific ST-T abnormalities exhibited a higher rate of unsatisfactory BP control  , and/or athletic ability.


Chronic arthropathy occurs in approximately two thirds of patients with hereditary haemochromatosis (HH). The aim was to study inflammatory and structural lesions in patients with HH with (HH-A) and without arthropathy (HH-WA) using ultrasonography.This was a cross-sectional study of 26 patients with HH-A, 24 with HH-WA and 37 with hand osteoarthritis (HOA). Clinical examination was performed in 68 joints, and we retrieved data on hand function, pain and global disease activity ), morning stiffness and ferritin levels. Standard x-ray and ultrasound were conducted in 36 joints , and we graded grey scale synovitis (GSS), power Doppler ultrasound (PD), osteophytes, erosions, tenosynovitis and cartilage damage semi-quantitatively in accordance with prior publications.Ultrasound revealed a high proportion of inflammatory changes in HH-A; GSS was found in 96.2% and PD signals in 80.8% of patients . The frequency of these findings was similar in HOA. Inflammation was also common in HH-WA, yielding GSS in 83.3% and PD signals in 50.0% of patients. Cartilage damage was most prominent in HH-A as compared to HH-WA and HOA . The prevalence and extent of erosions and osteophytes were similar in all groups. None of the ultrasound scores was associated with pain or function; GSS, PD, osteophyte and cartilage scores correlated with x-ray-verified structural damage.A high prevalence of ultrasound-verified inflammation and cartilage damage was found in HH-A, and to a lesser extent in HH-WA. These findings were associated with x-ray-verified damage but not with clinical scores of pain and function. HFE gene encoding a transmembranous glycoprotein leads to increased duodenal iron resorption despite full iron stores [HFE gene [Hereditary haemochromatosis (HH) is the most frequent autosomal-recessive inherited metabolic disorder with up to 0.5% homozygous mutations in Caucasian populations . A mutatn stores . The iron stores . DiagnosHFE gene , 4.Chronic arthropathy occurs in approximately two thirds of patients with HH . In the nd and 3rd metacarpophalangeal (MCP) joints; however, diagnosis may be difficult in cases of mild symptoms and/or involvement of other (particularly large) joints. Treatment of HH-A is challenging because regular phlebotomies do not lead to improvement in joint pain. Analgesics and/or non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used; however, the clinical efficacy of these drugs is variable and it is unknown whether treatment might modify the course of the disease [Diagnostic criteria for HH-A have not yet been established. The typical clinical picture is bony swelling and tenderness of the 2 disease .Imaging studies have rarely been performed in HH-A so far. Conventional radiography is the gold standard method for the detection of structural changes including joint space narrowing (JSN), osteophytes, calcium pyrophosphate deposition (CPPD) and/or subchondral osteosclerosis –9. MagneMusculoskeletal ultrasound has not yet been used to systematically study synovial and bony changes in patients with HH-A. The aim of this study was therefore to investigate inflammatory and structural abnormalities in patients with HH-A, HH without arthropathy (HH-WA) and hand OA (HOA) using ultrasonography.This was a cross-sectional study conducted at the Medical University of Graz, the Hanusch Hospital Vienna and the General Hospital Oberndorf . Approval by the respective institutional review boards and written informed consent of each patient were obtained.C282Y mutation or a compound mutation of the C282Y and H63D genes plus serological signs of iron overload at diagnosis: increased transferrin saturation  and either provisional  or overt iron overload (serum ferritin > 1000 ng/ml or hepatic iron overload on biopsy) independent of the presence of clinical symptoms related to iron overload [Patients with HH and hand OA (HOA) were recruited from clinical routine practice. HH was defined by a homozygous overload .HH-A was defined as HH plus pain in the hands (VAS > 10 mm and/or ≥ 1 tender joint) plus at least one radiographic change compatible with HH-A on hand x-rays , 12. TwoPatients with HOA were classified using the criteria of the American College of Rheumatology . All patAll patients underwent a structured history/chart review retrieving demographic data, medical history and current medication. Patients with HH were further questioned for the duration and frequency of phlebotomies, previous joint replacement and HH-related co-morbidities including hepatopathy, hypogonadism, cardiomyopathy, skin changes and diabetes mellitus.Clinical assessments included the duration of morning stiffness (minutes), number of tender joints (TJ) (68-joint count), swollen joints (SJ) (66-joint count), number of bony swollen joints  and distal interphalangeal joints (DIP), wrists, knees, and ankles), patient’s (PGA) and evaluator’s global assessment of disease activity (EGA), hand function and pain related to hands, hips, knees and ankles. Function, global and pain assessments were all recorded on a 100-mm VAS, with 0 = best, 100 = worst , 12.HFE gene mutation (C282Y and H63D) tests were also retrieved in order to classify the patients.Blood investigations included erythrocyte sedimentation rate  and C-reactive protein (CRP) (measured by nephelometry), blood cell count, liver enzymes, renal functional tests and iron status . The results of the Grey scale (GS) and power Doppler (PD) sonography were performed in 36 joints  by one of two rheumatologists (CDe and CDu) who were unaware of the clinical findings (but not blinded to diagnosis of HH versus HOA). We used a MyLab Twice ultrasound device  with two multi-frequence linear transducers . For GS imaging, parameters were adjusted to maximize the contrast between examined structures. PD settings were standardized accordingly: frequency 9.1  or 6.3 (large joints) MHz, pulse repetition frequency 750 Hz and medium persistence. The PD gain was optimized by increasing the gain until noise appeared and then reducing it just enough to suppress the noise .All ultrasonography comprised longitudinal and transverse scans in accordance with current guidelines and publications . GSS wasnd or 5th MCP joints) were counted as dorsal lesions. Osteophytes were further investigated by anterior scans of the hips and at the medial and lateral femorotibial spaces at the knees [Erosions  or osteophytes  were defined by a step-down or step-up contour defect, respectively that is visible in 2 perpendicular planes . At the he knees . Gradinghe knees  and was he knees ) with grhe knees ). ErosioCartilage was assessed at MCPs 2–5 on longitudinal scans with full flexion of the fingers, and at the knees on transverse scans with full flexion of the knees. Scoring was adapted from Filippucci et al. with a grading from 0 to 4, with 0 = no evidence of cartilage abnormalities, 1 = loss of sharpness of the superficial margin of the hyaline cartilage, 2 = partial thickness defect of the cartilage layer, 3 = full thickness defect of the cartilage layer with a normal subchondral bone profile and 4 = complete loss of the cartilage layer and subchondral bone involvement. CPPD deposits were defined as previously described and graded as 0 = absent or 1 = present . Sum scoPatients with HH underwent standard radiography of the hand and wrist, knee and ankle joints within 2 weeks of the study visit. All radiographs were assessed by one of two experienced radiologists (VT or SZ) and scored from 0 (no damage) to 6 (extensive damage) per joint as previously described . The totU test. Paired categorical data were analysed using the chi square test or Fisher’s exact test as appropriate. P values were not corrected for multiple testing.Statistical analysis was performed using SPSS (version 23.0). Descriptive statistics were used to summarize the data. The distribution of data was tested for normality using the Kolmogorov-Smirnov test. For continuous non-parametric data, we show the median and range whereas for parametric data, the mean and standard deviation are depicted. Comparisons between independent groups were conducted using the Mann-Whitney Inter-rater reliability of the ultrasound results was determined by serial blinded assessments of 10% of patients’ scans by two investigators (CDe and CDu) and using the intra-class correlation coefficient (ICC).There were 24 patients (27.6%) classified as HH-WA, 26 (29.9%) as HH-A and 37 (42.5%) as HOA. Clinical characteristics are detailed in Table p = 0.047; knee, 2 (8.0%) vs. 1 (4.3%) patient, p > 0.2). Patients with HH-A had higher levels of ankle pain as compared to patients with HOA, whereas the number of bony swollen joints and CRP levels were higher in patients with HOA than in patients with HH-A.As expected, there were more women in the HOA than in the HH groups, and disease duration was longer in patients with HH than in patients with HOA because most patients with HOA were diagnosed at the time of inclusion into the study. Patients with HH-A more commonly had liver fibrosis/cirrhosis than patients with HH-WA, whereas disease duration, ferritin levels at the time of the clinical visit and median duration (6 (0–26) vs. 6 (0–23) years, respectively) and the number of phlebotomies per year (2.5 0–12) vs. 3 (0–5), respectively) were similar in the HH-A and HH-WA groups. Clinical parameters of joint inflammation and function where higher in HH-A than in HH-WA by definition ; HH-WA, n = 13 (54.2%); HOA, n = 21 (56.8%); p = 0.036). Similarly, CPPD deposition was most common in patients with HH-A as compared to patients with HH-WA or HOA.Cartilage abnormalities were most common in patients with HH-A, which was mainly because of a high prevalence of cartilage damage in the MCP joints , p = 0.039 for comparison between HH-A and HH-WA and p = 0.097 between HOA and HH-WA). Erosion scores were similar in all groups ; HH-WA, 0 (0-5); HOA, 1 (0–7)), osteophyte scores were higher in HH-A (34 (4–75)) and HOA (36.0 (10–72)) than in HH-WA (23.5 (0–55); p = 0.024 for comparison between HH-A and HH-WA and p = 0.001 between HOA and HH-WA).As depicted in Fig. As detailed in Table p = 0.004) or HOA (2 (0–17), p < 0.001). Sub-analysis of scores of hands and knees revealed that the difference between groups was mainly related to cartilage damage at the MCP level ; HH-WA, 1.0 (0–24); HOA, 1.0 (0–15); p < 0.01 for comparison between HH-A and the other groups), whereas scores at the knees were comparable between groups ; HH-WA, 0 (0–6); HOA 0 (0–4)).Cartilage damage scores were highest in patients with HH-A ) as compared to patients with HH-WA (2.5 (0–25), coeff) 0.39, p = 0.05); however, neither of the ultrasound scores of inflammation or structural damage (nor their sub-scores for the hands) were linked with TJ, SJ, pain, global assessments or function.In patients with HH-A, we observed moderate correlation between the osteophyte score and the number of bony swollen joints  was observed, and GSS and PD scores at the hands correlated with cartilage damage .A negative association between the global PD score and ferritin levels (corrp = 0.007). Neither the global x-ray score nor the x-ray sub-score of hands was associated with pain, global assessments, function or ferritin levels.The median global x-ray score was higher in HH-A (15.5 (1–48)) than in patients with HH-WA (5.5 (0–29), coeff 0.70, p < 0.001 and corrcoeff 0.84, p < 0.001), GSS score , PD score  and cartilage score .In patients with HH-WA and HH-A, respectively, there was moderate to good correlation between the global x-ray score and the ultrasound osteophyte score  for the osteophyte score, 0.78 (95% CI 0.14–0.96) for the GSS score, 0.73 (95% CI 0.1–0.95) for the PD score and 0.96 (95% CI 0.81–0.99) for the cartilage score. Reliability testing for erosions and tenosynovitis was not possible because of the small number of abnormal findings in the subset of patients who were tested.In the present study, we demonstrate that ultrasound signs of inflammation and damage are common in patients with HH-A, particularly in the MCP joints. Albeit these findings had little impact on pain and function at the time of the clinical visit, they were associated with x-ray evidence of damage that in turn might lead to future disability , 23. PatAlthough arthropathy in patients with HH is common and joint symptoms are often disabling, systematic imaging studies investigating inflammatory and structural changes in HH-A have rarely been performed. A recent report highlighted the occurrence of PD synovitis in two cases of HH  and a smThe overall extent of inflammatory and structural changes was similar in HH-A and HOA, an observation that fits with a previous histological study comparing synovial tissues in patients with rheumatoid arthritis (RA), HH-A and OA, who were undergoing joint replacement surgery . In thatOne of the most intriguing findings of our study was the prominent cartilage damage in patients with HH-A, particularly in the MCP joints. In RA, a reduction in cartilage thickness has been shown to predict disability after 1–2 years, while it did not correlate with health assessment questionnaire (HAQ) scores at baseline , 30. We CPPD deposits were slightly more common in our cohort (37.5% in HH-WA and 61.5% in HH-A) than in previous cohorts of patients with HH (30–50%) , 36.The most important limitation of our study is the absence of a generally accepted definition of HH-A. While in some studies HH-A was defined as any joint pain associated with HH , we and Another limitation is that our patients with HH had long-standing disease and were on stable treatment, and that ferritin levels were thus within the normal range in almost all patients. Given that arthropathy in patients with HH has been linked with the duration and degree of iron overload , 39, it Further, the ultrasound investigators were not blinded to the diagnoses of HH versus HOA . This might have influenced the results; however, given the similar results between HH-A and HOA in regard to several aspects (and assuming that a lack of blinding would have increased the difference between these groups) and the good inter-reader reliability between investigators, we believe that the bias due to lack of blinding to diagnosis was negligible.The prevalence of inflammatory and structural changes is high in patients with HH-A. Overall inflammation and osteophytes were comparable between patients with HH-A and HOA, whereas cartilage damage was more prominent in the former compared to the latter group. Scores for inflammation and damage were highest in the MCPs in patients with HH-A, whereas in HOA there were higher osteophyte and GSS scores for the PIPs and DIPs.In patients with HH-WA, we observed a high prevalence of subclinical inflammation. These data demonstrate that HH-A is associated with synovial inflammation, contrasting with the long-believed hypothesis that joint pathology in HH is purely non-inflammatory.


Opsin proteins are seven transmembrane receptor proteins which detect light. Opsins can be classified into two types and share little sequence identity: type 1, typically found in bacteria, and type 2, primarily characterized in metazoa. The type 2 opsins (Rhodopsins) are a subfamily of G-protein coupled receptors (GPCRs), a large and diverse class of seven transmembrane proteins and are generally restricted to metazoan lineages. Fungi use light receptors including opsins to sense the environment and transduce signals for developmental or metabolic changes. Opsins characterized in the Dikarya (Ascomycetes and Basidiomycetes) are of the type 1 bacteriorhodopsin family but the early diverging fungal lineages have not been as well surveyed. We identified by sequence similarity a rhodopsin-like GPCR in genomes of early diverging chytrids and examined the structural characteristics of this protein to assess its likelihood to be homologous to animal rhodopsins and bind similar chromophores.Spizellomyces punctatus, a unicellular, flagellated species belonging to Chytridiomycota, one of the earliest diverging fungal lineages. We tested if the sequence and inferred structure were consistent with a solved crystal structure of a type 2 rhodopsin from the squid Todarodes pacificus.We used template-based structure modeling, automated ligand docking, and molecular modeling to assess the structural and binding properties of an identified opsin-like protein found in Spizellomyces opsin has structural characteristics consistent with functional animal type 2 rhodopsins and is capable of maintaining a stable structure when associated with the retinaldehyde chromophore, specifically the 9-cis-retinal isomer. Together, these results support further the homology of Spizellomyces opsins to animal type 2 rhodopsins.Our results indicate that the This represents the first test of structure/function relationship of a type 2 rhodopsin identified in early branching fungal lineages, and provides a foundation for future work exploring pathways and components of photoreception in early fungi. An organism experiences a multitude of environmental stimuli including chemical, gravity, the Earth’s magnetic field, pressure, and light. The biochemical ability to appropriately process and respond to these signals is a complex and involved task, and understanding the molecular mechanisms of these responses is an ongoing scientific challenge. The presence or absence of light is perhaps one of the easiest sources of stimuli to comprehend and observe. The daily cycles of sunlight due to the rotation of the planet has had such a profound influence on the development of life that it comes as no surprise to find some form of photoreception in nearly every organism on the planet. The widespread occurrence of such an ability, however varied in its implementation, speaks to its importance during the earliest stages of development of life.In Fungi, there are several classes of proteins capable of photoreception that function by different mechanisms of action and have varied structures, sensitivities, and specializations. These include blue light responsive white-collar complex, VIVID and cryptochrome photoreceptors, red light responsive phytochromes, and multi-wavelength light responsive opsins . The opsOpsins are part of the large G-protein coupled receptor (GPCR) superfamily, which has more than 800 distinct described members in humans . GPCR prcis-retinal is the most common chromophore observed in vertebrates and invertebrates, additional types are also found in nature. For example, 3,4-dehydroretinal is observed in fish, amphibians, and reptiles. Switching between the 11-cis- and 3,4-dehydro-chromophores can be employed as a light adaptation strategy in certain freshwater fish  is generated when an opsin apoprotein forms a covalent bond with a retinaldehyde chromophore via a Schiff-base linkage at a conserved lysine residue. While 11-ter fish . 3-hydrod 9-cis- . Previou pigment .cis-retinal to all-trans-retinal, which causes a conformational change in the protein structure of the receptor. Alternatively, the ion transporter rhodopsins (part of the “Type 1 opsins”) are activated by the photoisomerization of all-trans-retinal to 13-cis-retinal. These function as membrane channels and are typically used for light-driven membrane depolarization via proton or chloride ion pumping. Examples of this group can be found in bacteria, archaea, and eukaryotes, and include the bacterial sensory rhodopsins, channelrhodopsins, bacteriorhodopsins, halorhodopsins, and proteorhodopsins   .Batrachochytrium dendrobatidis genome revealed a surprising finding of a GPCR protein with similarity to the rhodopsin superfamily. Searches for additional genomes of early diverging fungi, including the saprotrophic chytrid Spizellomyces punctatus, revealed the presence of rhodopsin-like proteins in multiple zoosporic fungal lineages. The availability of these examples of opsin homologs in the deeply diverging fungal lineages suggested the shared ancestry of these light sensing receptors and the presence of this pathway in the fungal-animal ancestor . The amended protein sequence SPPG_00350T0L was used for subsequent analyses. The S. punctatus protein structure model was constructed using the I-TASSER server with the provided GPCR specific library , all-trans, 9-cis, 13-cis, 3,4-dehydro (A2), 3-hydroxy (A3), and 4-hydroxy (A4) used in the covalent docking screen. A covalent linkage was formed by manually specifying the presence of a bond between the terminal carbon atom in retinal and terminal nitrogen atom in the lysine side chain. The specific lysine predicted to be involved in Schiff-base linkage with the chromophore was inferred through multiple sequence alignment.Automated protein-ligand docking was accomplished using Autodock 4  and implTodarodes and Spizellomyces rhodopsin complexes were investigated using all-atom molecular dynamics simulations with the Amber14 suite of programs   . PotentiS. punctatus protein sequence using the I-TASSER website and GPCR specific database. Top Blastp hits of the S. punctatus protein to the PDB (as of 2016) include numerous opsin proteins, with the top scoring hit at 22.5% identity to a rhodopsin from Bos taurus RY and NPxxY motifs are both functionally conserved as 115ERY117 and 326NPVLF330 (pink). Two additional linkages are responsible for correct protein folding: a conserved disulfide bond between C110 and C187, and a conserved salt bridge between R117 and D190. S. punctatus model possesses both of these motifs as C91–C166 (yellow), and potentially R158–D169 (purple).The binding pocket comprises a number of hydrophobic residues which provide a sterically restrictive space in which the retinal ligand is situated (orange). The major functional residues in this group are the conserved lysine (cyan) and counterion (red) which facilitate proton transfer during photoisomerization. The ionic lock motif contains an (E/D)RY and NPxxY motif, which together act as a structural support which stabilizes the protein in the inactive (“dark”) state, and is broken upon receptor activation . In S. pS. punctatus homology model was assessed with Ramachandran plots  again suggested it was poorly modeled, with a global quality score of 0.42 and a range of local quality scores from −0.18 to 0.96  docking screen are provided in Rhodopsin functions through the use of a retinaldehyde chromophore. The most common chromophore observed in both invertebrates and vertebrates is 11--retinal . This ree tested  and all S. punctatus + 9-cis-retinal complex, molecular dynamics simulations on the opsin and unbound chromophore using AMBER 14 were performed and compared to that of the canonical squid + 11-cis-retinal complex. An overview of the potential energy of two systems during the 100 ns simulation is given in S. punctatus complex is much lower than that of the squid, both complexes are extremely stable over the long term. Using VMD to plot the RMSD relative to the averaged structure for both complexes also suggests they are stable. For both complexes, these results are given in S. punctatus complex fluctuates between 2.56 Å and 8.92 Å, with a mean and standard deviation of 4.67 ± 1.07 Å. Additionally, the per-residue RMSD for both structures remains low during the course of the simulation   . Fpockets pocket .S. punctatus simulation, the 9-cis-retinal ligand shifts approximately 1.6 Å inside the binding pocket of the model. A shift of approximately 1.8 Å by the functional nitrogen atom can be observed during the simulation. The ion lock distance (between E116 and R250) remained consistent, decreasing only slightly from 3.5 Å to 3.4 Å, while the disulfide bond distance (cysteine–cysteine link between C91 and C166) decreased from 5.4 Å to 3.7 Å  maintains its linear conformation. The T. pacificus ion lock and disulfide distances and orientations remained relatively unchanged, potentially due to the T. pacificus structure being closer to optimal conformation initially.During the course of the to 3.7 Å . During S. punctatus the measures of free energy using the structures from the end of the simulation (frame 3) were lowest when using 13-cis and 9-cis isomers of retinal , with the 11-cis isomer as the next lowest . For T. pacificus all isomers were relatively similarly low-scoring, although with slight increases using models near the end of the simulation.To assess any potential improvements in docking scores, revised covalent docking was performed using the structures resulting from the previously described simulations and the ligands presented in B. dendrobatidis and S. punctatus, we identified putative proteins homologous to metazoan Type 2 Rhodopsins. Rhodopsin functions as a photoreceptor via a well-defined interaction between a photon of light, a retinaldehyde chromophore , and the GPCR opsin protein in order to initiate a cellular response through intracellularly-coupled heterotrimeric G-proteins. There is evidence to suggest that the covalent bond architecture is not biochemically necessary in experimentally manipulated Type 1 opsins  are conserved, there are binding pocket residue differences between the squid and fungal structures which could account for the utilization of different chromophores. For example, fewer large hydrophobic residues in the fungal pocket might permit accommodation of different chromophores. Additionally, during the course of exploring why 11-cis-retinal was most often observed in mammalian systems, cis-retinal is only slightly less stable, and under certain conditions can in fact be more stable, than the 11-cis isomer. Our molecular docking results suggest that one preferential ordering of ligands would be: 9-cis > 13-cis > 11-cis > 4-hydroxy > all-trans > 3,4-dihydro- > 3-hydroxy-retinal. While a thorough treatment of the phylogenetic support for the shared ancestry of these proteins will be presented elsewhere , the functional relevance of such proteins remains to be explored. The S. punctatus + 9-cis-retinal complex after molecular dynamics simulations supports the hypothesis that this GPCR is a functional photoreceptor and provides a foundation for future work dealing with photoreception in early diverging fungi.In the present study we used 10.7717/peerj.3206/supp-1Figure S1S. punctatus iTasser homology model and B) T. pacificus rhodopsin x-ray crystal structure (PDB ID: 2Z73).A) Click here for additional data file.10.7717/peerj.3206/supp-2Figure S2T. pacificus crystal structure, (B) the S. punctatus homology model against the iTasser GPCR database, and (C) the S. punctatus homology model against the sensory rhodopsin II xray crystal structure from the archaeon Natronomonas pharaonis (PDBid 1H68). Far more residues have averaged scores below 0 in the S. punctatus model using 1H68 as the template than in the other two structures.(A) the Click here for additional data file.10.7717/peerj.3206/supp-3Figure S3T. pacificus crystal structure, (B) the S. punctatus homology model against the iTasser GPCR database, and (C) the S. punctatus homology model against the sensory rhodopsin II xray crystal structure from the archaeon Natronomonas pharaonis (PDBid 1H68). The global quality score for the fungal sequence against the sensory rhodopsin template was lower than that of the iTasser fungal model.(A) the Click here for additional data file.10.7717/peerj.3206/supp-4Table S1Table of similar protein sequences with structures in the PDB database to the SPPG_00350mod_Long .Click here for additional data file.10.7717/peerj.3206/supp-5Table S2The columns of this table: Rank, PDB ID, Protein sequence identity, Query protein coverage, Z-score, and sequence chain molecule descriptionClick here for additional data file.10.7717/peerj.3206/supp-6Table S3S. punctatus model or T. pacificus respectively.Panel 1 shows the energy and properties of the ligands, panels 2 and 3 shows the lowest energy terms for each retinal isomers from the covalent docking runs on the Click here for additional data file.10.7717/peerj.3206/supp-7Supplemental Information 1https://github.com/stajichlab/chytropsin.The sequence and report archive provides exonerate alignment results showing the original gene model aligned and post-correction of the genomic sequence. DNA and protein sequences of the corrected model are also provided. The parameters used to run exonerate are included in the report file. This is an archive of data available Click here for additional data file.